POS0411 COMPARISON OF MTX-POLYGLUTAMATE ACCUMULATION PROFILES IN PERIPHERAL BLOOD MONONUCLEAR CELLS AND ERYTHROCYTES DURING 6 MONTHS MTX-THERAPY IN THE METHOTREXATE MONITORING (MeMo) TRIAL
Authors: R. Hebing, M. Lin, E. Struys, S. Mahmoud, I. Muller, S. Heil, P. Griffioen, W. Lems, B. Van den Bemt, M. Nurmohamed, G. Jansen, R. De Jonge
Journal: Annals of the Rheumatic Diseases (2022)

Background Optimal dosing of methotrexate (MTX) in rheumatoid arthritis (RA) remains challenging. To this end, monitoring of intracellular MTX polyglutamates (MTX-PGs) in red blood cells (RBCs) has been investigated as a potential marker of MTX (non-)response, with contradictory results. As enucleated, non-proliferative cells, mature RBCs lack regulated folate metabolism and are devoid of folylpolyglutamate synthetase (FPGS) activity catalyzing the conversion of MTX to MTX-PGs. Therefore, it has been argued that analysis of MTX-PG in immune-effector cells, represented by peripheral blood mononuclear cells (PBMCs), would be more relevant. However, no prospective study has been performed measuring MTX-PG levels in PBMCs nor in comparison with RBCs. Objectives To investigate the pharmacokinetics of MTX-PG accumulation in RBCs and PBMCs in newly diagnosed RA patients in the early phase of MTX treatment. Methods In a clinical prospective cohort study (Methotrexate Monitoring (NTR7149)), RA patients were administered MTX op to 25 mg/week, as described before. (1) At 1, 2, 3 and 6 months after start of therapy, blood was collected and RBCs were isolated by centrifugation and PBMCs after Ficoll density gradient centrifugation. MTX-PG 1-6 concentrations in these cells were analyzed using a UPLC-MS/MS method with including custom-made stable isotopes of MTX-PG 1-6 as internal standards. (2) UPLC-MS/MS measurements for MTX-PG 1-6 were performed with a Waters Acquity BEH C18 column coupled to an AB Sciex 6500+ with the ESI operating on the positive mode. MTX dosing and concomitant treatments were in conformity with clinical practice. (3) Results 46 consecutive patients were included in this study; 76% female, mean age: 57.8 years, mean baseline DAS28-ESR: 3.5, as described before. (1) Mean dosage was 10.5 mg (SD: 1.5) at baseline, 16.3 mg (2.5) at month 1, 22.7 mg (4.5) at month 2, 19.5 mg (6.3) at month 3 and 19.1 mg (6.2) at month 6. MTX-PG accumulation in PBMCs and RBCs revealed a disparate profile in both MTX-PG distribution and absolute accumulation levels (Figure 1A/B). Remarkably, MTX-PG distribution in PBMCs was mainly composed of MTX-PG 1 (58%), and to a lesser extent MTX-PG 2 (27%) and MTX-PG 3 (15%). Longer chain MTX-PG 4-6 were also detectable in PBMCs, but at lower levels (mean: 4.0 – 6.7 fmol/10^6 cells) than MTX-PG 1-3 . Moreover, this MTX-PG distribution profile in PBMCs remained constant over a MTX therapy period of 6 months (Figure 1A). The RBC MTX-PG accumulation profile shows mainly MTX-PG 1 and lower levels of MTX-PG 2-6 at 1 month after the start of therapy. After 3 months of therapy, MTX-PG 3 is the main PG-moiety with also MTX-PG 4,5,6 being detected. This profile is largely similar after 6 months of therapy. With respect to total intracellular MTX-PG 1-6 accumulation, PBMCs had significantly (p&lt;0.001) 10-20-fold higher levels than RBCs at all analyzed time points (Figure 1A/B). Total MTX-PG 1-6 levels in RBCs and PBMCs at all time points were weakly correlated (r=0.41, p&lt;0.01) (Figure 1C). Figure 1. Individual MTX-PG concentrations in PBMCs (A) and RBCs (B) during the first 6 months of MTX administration (note the different scaling of the y-axes). At 6 months, 36 patients were still on MTX treatment. Panel (C): Spearman’s correlation plot of total MTX-PGs in RBCs versus PBMCs of all time points. The disparate MTX-PG accumulation and distribution profiles in PBMCs versus RBCs of RA patients may be associated with the shorter life span of PBMCs and the low FPGS activity in RBCs. (4) No significant relation between MTX-PGs and DAS28 was found (data not shown). Conclusion This study shows that in newly diagnosed RA patients starting MTX therapy, MTX-PG concentrations in PBMCs are significantly 10-20-fold higher than in RBCs over a period of 6 months, with a disparate MTX-PG distribution profile in PBMCs (highest: MTX-PG 1 ) than RBCs (highest: MTX-PG 3 ). References [1]RCF Hebing, Arthr Rheum (2021) [2]E den Boer, Anal Bioanal Chem (2013) [3]J Smolen, Ann Rheum Dis (2020) [4]IB Muller, Ther Drug Monit (2019) Acknowledgements Acknowledgements: We would like to thank all participating patients and Pfizer (grant 53233663 / WI230458), AmsterdamUMC (AI&amp;II extension grant) and NVKC (Noyons grant 2018) Disclosure of Interests Renske Hebing Grant/research support from: Pfizer (grant 53233663 / WI230458), NVKC (Netherlands Society for Clinical Chemistry, Noyons grant 2018) and AmsterdamUMC (extension grant), Marry Lin: None declared, Eduard Struys: None declared, Sohaila Mahmoud: None declared, Ittai Muller: None declared, Sandra Heil: None declared, Pieter Griffioen: None declared, WIllem Lems: None declared, Bart van den Bemt Speakers bureau: Pfizer, UCB, Sanofi-Aventis, Galapagos, Amgen and Eli Lilly, Michael Nurmohamed Grant/research support from: Pfizer grant 53233663 / WI230458, Gerrit Jansen: None declared, Robert De Jonge Grant/research support from: NVKC (Netherlands Society for Clinical Chemistry, Noyons grant)